1,2,3,6-tetrahydrophthalimide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Standard test method used (OECD test guideline) GLP statement

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,6-tetrahydrophthalimide

EC Number:

201-602-8

EC Name:

1,2,3,6-tetrahydrophthalimide

Cas Number:

85-40-5

Molecular formula:

C8H9NO2

IUPAC Name:

2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione

Details on test material:

- Name of test material (as cited in study report): P2008
- Analytical purity: 97.70%
- Impurities (identity and concentrations): Tetrahydrophthalic anhydride D3 - 0.02%; Tetrahydrophthalic anhydride D4 - 0.05%;
Tetrahydrophthalimide D3 - 1.48%; Tetrahydrophthalimide D4 isomer - 0.75%
- Lot/batch No.: 1801

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction prepared from the liver of rats treated with a single i.p. injection of Aroclor 1254 at 500 mg/kg. The S-9 mix used contained S-9 fraction and a cofactor solution

Test concentrations with justification for top dose:

Experiment 1: With and without S-9 mix: 8.0, 40, 200, 1000 and 5000 micrograms/plate
Experiment 2: With and without S-9 mix: 312.5, 625, 1250, 2500 and 5000 micrograms/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.1 mL per 2 mL medium)
- Justification for choice of solvent/vehicle: Commonly used agent allowing solubilisation of the test substance

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA100 and TA1535 in absence of S9

Controls 2

Positive control substance:

9-aminoacridine

Remarks:

for TA1538 in absence of S9

Controls 3

Positive control substance:

other: 4-Nitro-O-phenylenediamine

Remarks:

for TA1538 in absence of S9

Controls 4

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for TA98 in absence of S9

Controls 5

Positive control substance:

other: 2-Aminoanthracene

Remarks:

for TA1535 in presence of S9

Controls 6

Positive control substance:

9-aminoacridine

Remarks:

for TA98, TA100, TA1537 and TA1538 in presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Exposure duration: 48 hours


SELECTION AGENT (mutation assays): Histidine deficient medium


NUMBER OF REPLICATIONS: 3



DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Other - thinning of background lawn

Evaluation criteria:

Positive response: Substance induces a dose-related and statistically significant increase mutation rate in one of more strains of bacteria in the
presence and/or absence of S9 in both experiments at sub-toxic concentrations
Negative response: Number of induced revertants, compared to spontaneous revertants, less than two-fold at each concentration examined

Statistics:

UKEMS recommended methods

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results
Experiment 1: Without S9 mix
	Concentration	Number of revertants (colonies/plate)
Treatment	(microgram/plate)	Base-pair substitution type			Frameshift type
		TA100	TA1535	TA1538	TA98		TA1537
	0	139.0	12.7	21.0	22.7		13.7
	8	125.3	18.0	22.7	27.3		12.3
Test	40	118.0	12.0	25.3	19.3		13.7
substance	200	117.7	17.7	25.3	24.0		12.3
	1000	118.7	15.3	22.0	23.7		14.0
	5000	123.3	16.0	20.3	25.0		13.0
	N-ethyl-N’-nitro-N-nitrosoguanidine	536.3	109.7	-	-		-
Positive	4-nitro-O-phenylenediamine	-	-	412.0	-		-
controls	4-nitroquinoline N-oxide	-	-	-	184.0		-
	9-aminoacridine	-	-	-	-		352.0
Experiment 1: With S9 mix
	Concentration	Number of revertants (colonies/plate)
Treatment	(microgram/plate)	Base-pair substitution type			Frameshift type
		TA100	TA1535	TA1538	TA98		TA1537
	0	147.3	19.0	23.3	36.0		11.0
	8	137.3	19.7	27.3	31.7		9.0
Test	40	144.0	18.7	25.3	31.7		12.0
substance	200	133.3	20.7	25.0	34.0		10.7
	1000	140.0	22.7	22.0	34.7		12.3
	5000	132.0	14.7	25.0	29.7		14.3
Positive	Benzo(a)pyrene	517.0	-	225.7	525.0		179.7
controls	2-aminoantracene	-	236.3	-	-		-
Experiment 2: Without S9 mix
	Concentration	Number of revertants (colonies/plate)
Treatment	(microgram/plate)	Base-pair substitution type			Frameshift type
		TA100	TA1535	TA1538	TA98		TA1537
	0	124.7	14.7	13.0	18.0		11.3
	312.5	103.3	13.7	15.0	19.0		13.7
Test	625	101.0	14.0	16.7	19.3		10.3
substance	1250	102.7	13.3	10.3	19.0		11.7
	2500	94.7	13.7	13.0	19.3		10.7
	5000	105.7	12.3	11.3	20.3		13.7
	N-ethyl-N’-nitro-N-nitrosoguanidine	607.3	207.7	-	-		-
Positive	4-nitro-O-phenylenediamine	-	-	406.0	-		-
controls	4-nitroquinoline N-oxide	-	-	-	191.3		-
	9-aminoacridine	-	-	-	-		791.3
Experiment 2: With S9 mix
	Concentration	Number of revertants (colonies/plate)
Treatment	(microgram/plate)	Base-pair substitution type			Frameshift type
		TA100	TA1535	TA1538	TA98		TA1537
	0	128.7	15.7	21.3	23.3		13.0
	312.5	129.0	17.0	21.3	24.3		12.3
Test	625	121.3	16.7	22.3	18.7		13.0
substance	1250	120.3	16.3	23.0	21.0		14.3
	2500	117.7	19.3	22.3	24.0		12.7
	5000	115.3	19.0	17.7	26.3		13.3
Positive	Benzo(a)pyrene	531.7	-	291.0	445.3		133.3
controls	2-aminoantracene	-	361.3	-	-		-